Aptamers are nucleic acid-based probe molecules that provide similar affinity and specificity as antibodies towards protein targets. Aptamers have found utility in a wide variety of diagnostic and therapeutic applications. In 2011, AptaMatrix, In. helped pioneer a new method for discovering aptamer probes, called Acyclic Identification of Aptamer (AIA) that leveraged the sequencing capacity of Illumina high-throughput sequencing platforms. This Phase I SBIR project is focused on further improving the AIA method by integrating a post-sequencing, Illumina protein screening step (secondary screen) into the AIA workflow. We have established three project aims to successfully complete this project: (1) within the Illumina flowcell, test/optimize a novel method for generating truncated single-stranded DNA (ssDNA) clusters needed for optimal target protein binding on the sequencing system, (2) integrate and optimize established protocols and software needed to image DNA-protein binding, and (3) validate the complete method by monitoring DNA-protein binding affinities using a well characterised thrombin-thrombin binding aptamer (TBA), DNA library system. Completion of this project will establish a robust and highly valuable platform for rapid and highly efficient aptamer discovery and assessment. In Phase II we plan to scale up this method on our GAiix platform to offer rapid aptamer discovery services, and consider purchasing a next generation Illumina sequencing platform to implement protein imaging capabilities, to accommodate higher sample throughput at a lower operational cost.